This invention relates to a novel integrin alpha chain; functionally-equivalent peptide fragments and analogs thereof; oligonucleotides encoding the peptide fragments and analogs; vectors containing and cell lines expressing the novel peptides; and methods for using the peptide fragments, analogs and oligonucleotides.
The integrin mediated adhesive interactions of cells with other cells and between cells and the extracellular matrix are believed to play critical roles in a wide variety of processes including, for example, modulation of the immune system, regulation of developmental processes and tumor progression and metastasis. These molecules also transduce information from the extracellular to the intracellular environment through poorly understood signalling mechanisms. The integrins represent one of the best characterized superfamilies of adhesion receptors. Integrins are glycoprotein heterodimers which contain a non-covalently associated xcex1 and xcex2 subunit. Integrin subunits are transmembrane proteins which contain an extracellular domain for interacting with an extracellular matrix or cellular component, a transmembrane domain spanning the cell membrane and a cytoplasmic domain for interacting with one or more cytoskeletal components.
There are fourteen known xcex1 subunits and eight known xcex2 subunits which can pair to form at least twenty different integrin molecules. Several distinct integrin xcex1 chains are capable of pairing with one type of xcex2 chain to form a xcex2 chain subfamily. Thus, for example, the xcex21 subfamily includes seven members (also known as the VLA proteins: xcex11xcex21-xcex17xcex21); the xcex22 subfamily includes three members (the leukocyte cell adhesion molecules or LeuCAMs: xcex1Lxcex22 or LFA-1, xcex1Mxcex22 or Mac-1 and xcex1xxcex22 or p150,95) and the xcex23 subfamily includes two members (avxcex23, xcex1IIbxcex23). In some instances, an xcex1 chain may pair with more than one xcex2 chain, e.g., xcex14 can pair with xcex21 or xcex27.
The integrin xcex1 chains have in common a seven-fold repeated amino acid motif, of which the last three or four motifs include divalent cation binding sites. All known xcex1 chains have been divided into one of two structural groups on the basis of amino acid sequence homology and the presence or absence of two structural features (described below).
The first group of xcex1 chains contains a proteolytic cleavage site located in the extracellular domain, proximal to the transmembrane region. Post-translational cleavage of the xcex1 chain precursor yields two fragments which (with one exception) remain associated by a disulfide linkage. The smaller fragment includes a short portion of the extracellular domain, the transmembrane and the cytoplasmic domains. The larger fragment contains the major portion of the xcex1 chain extracellular domain. This group of post-translationally cleaved integrin xcex1 subunits includes xcex13, xcex14, xcex15, xcex16 (formerly called xcex1E), xcex1v and xcex1IIb, although the xcex14 molecule is a more distant member of the group since it is less similar to the other cleaved integrin xcex1 subunits based upon homology analysis, is cleaved near its mid-point to yield two fragments of nearly equal size and further, because the fragments are not disulfide linked (Teixido, J. et al., (1992) J. Biol. Chem. 267, 1786-1791; Rubio, M. et al., (1992) Eur. J. Immunol. 22, 1099-1102).
Members of the second group of integrin a subunits do not include the above-described proteolytic cleavage site. Moreover, the second group of a subunits is characterized by the presence of an additional region known as the xe2x80x9cIxe2x80x9d (inserted) domain. Homologous I domains have been identified in complement factors B and C2, von Willebrand""s factor, cartilage matrix glycoprotein and collagen type VI.
The importance of integrins with respect to modulation of the immune system is illustrated by the condition, leukocyte adhesion deficiency (LAD), a disorder that is characterized by profound immunodeficiency. Individuals afflicted with LAD are unable to express the xcex22 integrin subfamily (Hogg, N. (1989) Immunol. Today 10, 111-114). Thus, while it has been known for some time that integrins and other adhesion molecules function in immune system modulation, e.g., by playing a role in the adhesion of peripheral lymphocytes to endothelium and in homing to lymph nodes. However, relatively little is known about the molecules that function in the mucosal immune system, a subset of the general immune system which includes the lymphocytes which populate the gastrointestinal, genito-urinary and respiratory tracts, and the mammary glands. In particular, little is known about the molecules which function in mucosal lymphocyte homing. (see, Cepek, K. et al., (1993) J. Immunol. 150, 3459-3470 and references cited therein).
Recently, we described a novel integrin heterodimer that is expressed on intra-epithelial T lymphocytes (iIEL), i.e., the population of T lymphocytes located along the baso-lateral surfaces of the epithelial cells which line the mucosa, adjacent to the epithelial cell basement membrane. (Parker, C. M. et al., (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1924-1928). Originally defined by an antibody which recognizes the human mucosal lymphocyte 1 antigen (HML-1), the novel integrin is present on  greater than 90% of intestinal IEL (iIEL) and on approximately 40% of lamina propria T lymphocytes (which lie between the epithelial basement membrane and the muscularis mucosae) (Cerf-Bensussan, N. et al., (1987) Eur. J. Immunol. 17, 1279-1285). The HML-1 antigen contains a novel a chain (designated xcex1E, for xe2x80x9cepithelial associatedxe2x80x9d) associated with a xcex27 chain (Parker, C. M. et al., (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1924-1928). Although the HML-1 xcex27 chain has been cloned (Yuan, Q. A. et al., (1990) Int. Immunol. 2, 1097-1108; Erle, D. J. et al., (1991) J. Biol. Chem. 266, 11009-11016), little is known about the primary structure of the xcex1E chain.
Cloning of the xcex1E chain has proven to be problematic because of the difficulty in obtaining an adequate number of intra-epithelial cells from which the xcex1E chain could be purified and sequenced. In addition, the relatively large length of the xcex1E gene has hampered cloning efforts because of the propensity to lose the 5xe2x80x2 portion of relatively long genes during cDNA synthesis.
The cDNA sequence and derived amino acid sequence (Sequence I.D. Nos. 1 and 2, Genbank Accession Number L25851) for the HML-1 xcex1 chain are disclosed herein. The xcex1E chain was purified from a hairy cell leukemia spleen lysate and the N-termini of the purified protein fragments (25 kDa and 150 kDa) were sequenced to prepare degenerate oligonucleotide probes for screening a cDNA library. Surprisingly, in view of its resemblance to other integrin xcex1 subunits in overall amino acid sequence, xcex1E contains a region of 55 amino acids (referred to herein as the xe2x80x9cXxe2x80x9d (extra) domain or Sequence I.D. No. 4, encoded by Sequence I.D. No. 3) located N-terminal to the I domain. This region is not present in any other integrin.
The X domain contains two unique structural features: (1) an internal proteolytic cleavage site (between amino acids 160 and 161 of Sequence I.D. No. 1) followed by (2) a highly charged region of 18 consecutive amino acids (amino acids 163-180). Upon in vivo proteolytic cleavage of the xcex1E chain, two fragments are formed: a 26 kD fragment having a C-terminus which contains the 34 amino acids of the X domain N-terminus and a 150 kD fragment having an N-terminus which contains the 21 amino acids of the X domain C-terminus. The 21 amino acid portion of the X domain (referred to hereinafter as Sequence I.D. No. 5) includes the highly charged region of 18 consecutive aminoacids. The 34 amino acid portion of the X-domain N-terminus is referred to hereinafter as Sequence I.D. No. 6. These unique structural features of the X domain, coupled with the discovery that high levels of the mRNA for xcex1E (and for xcex27, with which it associates to form the integrin xcex1Excex27) are restricted to mucosal lymphocytes, suggested to us that the xcex1E subunit likely plays a unique role in the localization and/or site specific functions of intra-epithelial T-lymphocytes.
One aspect of the invention is directed to isolated peptides which inhibit the in vivo and in vitro function of the xcex1E subunit. The peptides have sequences which are related to, or derived from, the amino acid sequence of the above-described X domain (Sequence I.D. No. 4), e.g., Sequence I.D. Nos. 5, 6 and functionally equivalent peptide analogs of the foregoing peptides.
According to another aspect of the invention, a method for selecting a functionally equivalent peptide analog of Sequence I.D. No. 4 is provided. The method includes providing a peptide analog of Sequence I.D. No. 4 and determining whether the peptide analog inhibits adhesion between a human mucosal lymphocyte-1 antigen and an epithelial cell in vitro. Preferably, the peptide analogs are between about four and about twenty amino acids in length. More preferably, the peptide analogs are between about four and about ten amino acids in length. Exemplary peptide analogs are disclosed in Sequence I.D. Nos. 8 through 25, inclusive.
According to yet another aspect of the invention, a method for screening a molecular library to identify lead compounds which inhibit the in vivo activity of the integrin xcex1E chain is provided. The method includes determining whether the molecular library contains a compound which inhibits adhesion between a human mucosal lymphocyte-1 antigen and an epithelial cell in vitro. Also provided is a competitive binding assay method for identifying lead compounds which mimic the ligand binding site of the integrin xcex1E chain. The method involves determining whether the library contains a molecule which competitively inhibits the binding of the xcex1E chain (or a functionally equivalent peptide fragment or analog thereof) to an antibody which specifically recognizes the ligand binding site of the xcex1E chain.
According to another aspect of the invention, a pharmaceutical composition is provided. The composition includes a therapeutically effective amount of one or more of the above-identified isolated peptides (e.g., Sequence I.D. No. 4, a fragment or functionally equivalent peptide analog thereof) and a pharmaceutically acceptable carrier therefor, optionally, the composition further includes the xcex27 chain or a portion thereof in association with the above-identified isolated peptide. Alternatively, the pharmaceutical composition includes a therapeutically effective amount of one or more isolated oligonucleotides and a pharmaceutically acceptable carrier therefor.
According to still another aspect of the invention, a support having a biologically active surface which exhibits cell attachment activity is provided. The support includes a surface to which is attached one or more of the above-disclosed peptides. Exemplary supports include a prosthesis device (e.g., a vascular graft, a percutaneous device) and an affinity matrix (e.g., for isolating the ligand(s) of the xcex1E subunit).
According to another aspect of the invention, a method for isolating a cell surface ligand of the integrin xcex1E chain is provided. The method involves coupling Sequence I.D. No. 4, or a functionally equivalent fragment or peptide analog thereof (e.g., Sequence I.D. Nos. 5-6 and 8-25), to an inert support and isolating the cell surface ligand using affinity chromatography.
According to yet another aspect of the invention, antibodies to the isolated peptides or isolated oligonucleotides are provided. The antibodies are useful for blocking a functional activity of intra-epithelial T lymphocytes, such as an in vivo functional activity (e.g., localization of the T lymphocytes) or an in vitro functional activity (e.g., adhesion of intra-epithelial T lymphocytes to an epithelial cell monolayer as determined in an adhesion assay). Accordingly, the antibodies are useful as reagents in screening assays to identify lead compounds that are present in molecularly diverse libraries or other mixtures.
According to yet another aspect of the invention, an isolated oligonucleotide is provided. The isolated oligonucleotide encodes a peptide selected from the group consisting of Sequence I.D. Nos. 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25. In a particularly preferred embodiment, the isolated oligonucleotide comprises Sequence I.D. No. 7, an oligonucleotide which encodes Sequence I.D. No. 6.
Also provided is an isolated oligonucleotide that is capable of hybridizing under stringent conditions (defined below) to the nucleotide sequence residing between about position 558 and about position 659 of Sequence I.D. No. 1. This region of the xcex1E cDNA encodes Sequence I.D. No. 6 (i.e., the relatively uncharged proteolytic cleavage fragment of the X domain). In a preferred embodiment, the isolated oligonucleotide is at least about 80% homologous with the above-recited nucleotide region of Sequence I.D. No. 1. More preferably, the isolated oligonucleotide is 100% homologous with the nucleotide sequence residing between position 558 and position 659 (referred to hereinafter as Sequence I.D. No. 7) inclusive of the xcex1E cDNA.
According to still another aspect of the invention, an antisense oligonucleotide capable of hybridizing under stringent conditions to the above-described isolated oligonucleotide is provided. The antisense oligonucleotide is capable of hybridizing to a unique fragment (defined below) of the naturally-occurring DNA or mRNA encoding the xcex1E subunit. Accordingly, delivery of an antisense oligonucleotide to intra-epithelial lymphocytes in vivo inhibits localization of the lymphocytes by base-pairing with the DNA (or RNA) encoding a unique fragment of the xcex1E nucleic acid, thereby preventing transcription (or translation) of the xcex1E subunit.
According to yet other aspects of the invention, a recombinant expression vector comprising at least one strand of the above-disclosed isolated oligonuclsotide and a cell line transfected with the recombinant expression vector are provided. Preferably, the oligonucleotide is operatively joined to at least one regulatory sequence, for example, a promoter or enhancer sequence. Suitable cell lines include mammalian cells; bacterial cells; insect cells and various yeast strains.
These and other aspects of the invention, as well as various advantages and utilities, will be more apparent with reference to the detailed description of the preferred embodiments and in the accompanying drawings.